ARTICLES
NEWS IN PHYSIOLOGICAL SCIENCES
Fusion Pore in Live Cells
Bhanu P. Jena
Departments of Physiology and Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201
Earlier electrophysiological measurements on live secretory cells suggested the presence of fusion
pores at the plasma membrane, where secretory vesicles fuse to release vesicular contents.
Recent studies using atomic force microscopy demonstrate for the first time the presence of the
fusion pore and reveal its morphology and dynamics at near-nanometer resolution and in
real time.
T
he fusion of membrane-bound secretory vesicles at the cell
plasma membrane and consequent expulsion of vesicular
contents is a fundamental cellular process regulating basic
physiological functions such as neurotransmission, enzyme
secretion, and hormone release. Secretory vesicles dock and
fuse at specific plasma membrane locations following secretory stimuli. Earlier electrophysiological studies on mast cells
suggested the existence of “fusion pores” at the cell plasma
membrane, which become continuous with the secretory vesicle membrane following stimulation of secretion (13). By
using atomic force microscopy (AFM), the existence of the
fusion pore was confirmed, and its structure and dynamics in
both exocrine (16, 19) and neuroendocrine cells (7, 9) were
determined at near-nanometer resolution and in real time.
Why had this new cellular structure (the fusion pore) eluded
visualization in live cells for so long? The answer lies simply in
the resolution limit of the light microscope, which is dependent on the wavelength of the light used, and hence the
resolving power would be at best 300
400 nm. The recently
discovered fusion pore in live cells is cone shaped, measuring
100
150 nm at its wide end and 15
30 nm in relative depth.
As a result, it had evaded visual detection. With the development of AFM (4) and its improved capabilities to image biological samples at near-nanometer resolution, cellular structures such as the fusion pore and its dynamics could be examined at nanometer resolution and in real time (1, 2, 18). In
AFM, a probe tip microfabricated from silicon or silicon
nitride and mounted on a cantilever spring is used to scan the
surface of the sample at a constant force (1). Either the probe
or the sample can be precisely moved in a raster pattern by
using an xyz piezo tube to scan the surface of the sample (5).
The deflection of the cantilever measured optically is used to
generate an isoforce relief map of the sample (2). AFM therefore allows imaging at nanometer resolution and in real time
of live cells, subcellular structures, or single molecules submerged in physiological buffer solutions. Structure and
dynamics of the fusion pore at nanometer resolution is just the
first of many structures waiting to be identified in the living
cell. This finding has opened the window to a new under0886-1714/02 5.00 © 2002 Int. Union Physiol. Sci./Am. Physiol. Soc.
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standing of the workings of a living cell. In this review, the
structure and dynamics of the fusion pore in live cells, as
determined by using the AFM, is presented.
New cellular structure
Isolated live pancreatic acinar cells in physiological buffer,
when imaged by using AFM (8, 19), reveal at the apical
plasma membrane a group of circular “pits” measuring 0.4
1.2 2m in diameter, punctuated by smaller “depressions”
within. Each depression averages ~100
150 nm in diameter
(Fig. 1), and typically three to four depressions are located
within a pit. The basolateral membranes of acinar cells are,
however, devoid of either pits or depressions. High-resolution
AFM images of depressions in live cells further reveal a coneshaped morphology (Fig. 2). The depth of each depression
cone measures ~15
30 nm. Similarly, both growth hormone
(GH)-secreting cells of the pituitary gland and the chromaffin
cell also possess pits and depression structures on their plasma
membranes (7, 9), suggesting the universal presence of fusion
pores in secretory cells.
Regulation and dynamics of depressions
Exposure of pancreatic acinar cells to a secretagogue
(mastoparan) results in a time-dependent increase (20
35%)
in depression diameter, followed by a return to resting size following completion of secretion (Fig. 3). However, no demonstrable change in pit size is detected during this time. Enlargement of depression diameter and an increase in its relative
depth following exposure to secretagogues correlated with
increased secretion. Exposure of pancreatic acinar cells to
cytochalasin B, a fungal toxin that inhibits actin polymerization, results in 15
20% decrease in depression size and a consequent 50
60% loss in secretagogue-induced secretion.
Results from these studies suggested that depressions are the
fusion pores in pancreatic acinar cells. Furthermore, these
studies demonstrated the involvement of actin in regulation of
the structure and function of depressions.
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222, 2002;
10.1152/nips.01394.2002
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FIGURE 1. Topology of the apical cell surface of an isolated live pancreatic
acinar cell, observed by using atomic force microscopy. Scattered pits (one
shown with dotted outline) and depressions (arrowheads) are identified. See
Schneider et al. (19).
Analogous to pancreatic acinar cells, examination of resting
GH-secreting cells of the pituitary (7) and chromaffin cells of
the adrenal medulla (9) also reveal the presence of pits and
depressions on the cell plasma membrane. Depressions in
resting GH cells measure 154 H 4.5 nm (mean ± SE) in diameter. Exposure of the GH cell to a secretagogue resulted in a
40% increase (215 ± 4.6 nm; P < 0.01) in depression diameter but no appreciable change in pit size.
Depressions are fusion pores
Enlargement of depression diameter following exposure of
FIGURE 2. Nanometer resolution of a single depression or fusion pore in a
live pancreatic acinar cell. Note the cone-shaped fusion pore, with a 100- to
150-nm opening. See Cho et al. (8).
acinar cells to a secretagogue correlated with increased secretion. Additionally, actin-depolymerizing agents known to
inhibit secretion (19) resulted in decreased depression size
and accompanied loss in secretion. These studies suggested
depressions to be the fusion pores. However, a more direct
determination of the function of depressions was required.
Combining the use of a gold-conjugated antibody to a specific
vesicular secretory protein with AFM provides the means to
determine if secretion occurs at depressions (8, 7). The membrane-bound secretory vesicles in exocrine pancreas contain
the starch-digesting enzyme amylase. By using amylase-specific immunogold AFM studies, localization of amylase at
depressions following stimulation of secretion was demon-
FIGURE 3. Dynamics of depressions following stimulation of secretion. Top: a number of depressions within a pit in a live pancreatic acinar cell. The scan line
across 3 depressions is represented graphically at middle and defines the diameter and relative depth of the depressions; the middle depression is represented
by red arrowheads. Bottom: percentage of total cellular amylase release in the presence and absence of the secretagogue Mas7. Notice an increase in the diameter and depth of depressions, correlating with an increase in total cellular amylase release at 5 min after stimulation of secretion. At 30 min after stimulation of
secretion, there is a decrease in diameter and depth of depressions, with no further increase in amylase release over the 5-min time point. No significant increase
in amylase secretion or depression diameter were observed in resting acini or those exposed to the nonstimulatory mastoparan analog Mas17. See Schneider et
al. (19).
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FIGURE 4. Fusion pores (depressions) dilate to allow expulsion of vesicular contents. A
C: atomic force microscopy (AFM) micrographs of pits (dashed circle)
and depressions (red circles) showing enlargement of depressions following stimulation of secretion (from A to B). Exposure of live cells to gold-conjugated to
amylase antibody results in specific localization of gold to secretory sites (C). Note the localization of amylase-specific immunogold at the edge of depressions.
D: AFM micrograph of pits and depressions with immunogold localization is also demonstrated in cells immunolabeled and then fixed. See Cho et al. (8).
strated (Fig. 4) (8). These studies confirm depressions to be the
fusion pores in pancreatic acinar cells, where membranebound secretory vesicles dock and fuse to release vesicular
contents (Fig. 5). Similarly in somatotrophs of the pituitary,
gold-tagged GH-specific antibodies were found to be selectively localized at depressions following stimulation of secretion (7), again confirming depressions to be fusion pores.
Composition of the fusion pore
Although the molecular composition of the fusion pore
(depression) remains to be established, our studies on the role
of actin in the regulation of depression structure and dynam-
ics clearly suggests actin to be a major component of the
fusion pore complex. Target membrane proteins SNAP25 and
syntaxin (t-SNARE) and secretory vesicle-associated membrane protein (v-SNARE) are part of a conserved protein complex involved in fusion of opposing bilayers (17, 20). Since
membrane-bound secretory vesicles dock and fuse at depressions to release vesicular contents, it is reasonable to suggest
that plasma membrane-associated t-SNAREs are part of the
fusion pore complex. In the past decade, a number of studies
demonstrated the involvement of cytoskeletal proteins in exocytosis, some directly interacting with SNAREs (3, 8, 10, 11,
14, 15). Actin and microtubule-based cytoskeleton have been
implicated in intracellular vesicle traffic (11). Fodrin, which
FIGURE 5. Schematic diagram of the plasma membrane (PM) showing a pit with smaller depressions within. Depressions are the fusion pores where membranebound secretory vesicles (SV) dock and fuse to release vesicular contents.
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was previously implicated in exocytosis (3), has recently been
shown to directly interact with SNAREs (14). Recent studies
demonstrate that ,-fodrin regulates exocytosis through its
interaction with the syntaxin family of proteins (14). The
COOH-terminal coiled coil region of syntaxin interacts with
,-fodrin, a major component of the submembranous
cytoskeleton. Similarly, vimentin filaments interact with
SNAP23/25 and control the availability of free SNAP23/25 for
assembly of the SNARE complex (10). Additionally, our recent
studies (unpublished observations) demonstrate a direct interaction between actin and SNAREs. Results from these studies
suggest that vimentin, ,-fodrin, actin, and SNAREs may all be
part of the fusion pore complex. However, purification and
further biochemical characterization of the fusion pore are
required to determine its composition. Additional proteins
such as v-SNARE (VAMP or synaptobrevin), synaptophysin,
and myosin may associate when the fusion pore establishes
continuity with the secretory vesicle membrane. The globular
tail domain of myosin V is its binding site, and VAMP is bound
to myosin V in a calcium-independent manner (15). Further
interaction of myosin V with syntaxin requires calcium and
calmodulin. Studies suggest that VAMP acts as a myosin V
receptor on secretory vesicles and regulates formation of the
SNARE complex (15). Furthermore, interaction of VAMP with
synaptophysin and myosin V has been demonstrated (8).
Perspectives and conclusion
Fusion pores or depressions in pancreatic acinar or GHsecreting cells are cone-shaped structures at the plasma membrane, with a 100- to 150-nm-diameter opening. Membranebound secretory vesicles ranging in size from 0.2 to 1.2 2m in
diameter dock and fuse at depressions to release vesicular
contents. Following fusion of secretory vesicles at depressions,
only a 20
35% increase in depression diameter is demonstrated. It is therefore reasonable to conclude that secretory
vesicles “transiently” dock and fuse at depressions. In contrast
to accepted belief, if secretory vesicles were to completely
incorporate at depressions, the fusion pore would distend
much wider than what is observed. Furthermore, if secretory
vesicles were to completely fuse at the plasma membrane,
there would be a loss in vesicle number following secretion.
Examination of secretory vesicles within cells before and after
secretion demonstrates that, although the total number of
secretory vesicles remains unchanged following secretion, the
number of empty and partially empty vesicles increases significantly, supporting the occurrence of transient fusion (6). Earlier studies on mast cells also demonstrated an increase in the
number of spent and partially spent vesicles following stimulation of secretion, without any demonstrable increase in cell
size (12). Other supporting evidence favoring transient fusion
is the presence of neurotransmitter transporters at the synaptic
vesicle membrane. These vesicle-associated transporters
would be of little use if vesicles were to fuse completely at the
plasma membrane to be endocytosed at a later time. Although
the fusion of secretory vesicles at the cell plasma membrane
occurs transiently, complete incorporation of membrane at the
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cell plasma membrane takes place when cells need to incorporate signaling molecules like receptors, second messengers,
and ion channels.
I wish to thank Sang-Joon Cho for help in preparation of the figures and
David M. Lawson for valuable comments and suggestions.
Studies performed in my laboratory were supported by Grants DK-56212
and NS-39918 from the National Institutes of Health.
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